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Bacillus subtilis engineering bacteria with high yield of acetylglucosamine and application thereof

A technology of Bacillus subtilis and glucosamine, applied in the field of genetic engineering, can solve the problems of severe environmental pollution, unsuitable for people with seafood allergies, etc., and achieve the effect of simple construction method, convenient use, and good application prospects

Active Publication Date: 2013-04-24
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, acetyl glucosamine is mainly produced by acid-decomposing chitin in shrimp shells or crab shells. The waste liquid produced by this method is relatively serious for environmental pollution, and the obtained products are easy to cause allergic reactions, so it is not suitable for people with seafood allergies.

Method used

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  • Bacillus subtilis engineering bacteria with high yield of acetylglucosamine and application thereof
  • Bacillus subtilis engineering bacteria with high yield of acetylglucosamine and application thereof
  • Bacillus subtilis engineering bacteria with high yield of acetylglucosamine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Knockout of the gene encoding acetylglucosamine transporter (nagP)

[0017] According to the upstream and downstream sequences of the acetylglucosamine transporter gene (nagP) of Bacillus subtilis (Bacillus subtilis168, purchased from the American Type Microorganism Collection, ATCC No. 27370) published on NCBI, the homology arm amplification primers for the knockout frame were designed, The upstream and downstream primers of the left arm are: nagP-L-F: 5'-AATGAGATGCCTGTGTCGGAATA-3' and nagP-L-R:

[0018] 5'-TCCTGTGTGAAATTGTTATCCGCTCATCCACTCTCCAAACGAGTTGATAC-3'; the upper and lower primers of the right arm are: nagP-R-F:

[0019] 5'-ACGTCGTGACTGGGAAAACCCTGGCCGCGGTCTTAACCGGGTTA-3' and nagP-R-R:

[0020] 5'-TACGACAACGCCCAGCTTC-3'. The left and right arms of the nagP knockout box were amplified from the Bacillus subtilis168 genome using the above primers. According to the p7Z6 plasmid sequence published on NCBI (Nanjing Agricultural University, gifted by Dr. Y...

Embodiment 2

[0024] Example 2 Knockout of the gene encoding acetylglucosamine deacetylase (nagA) and the gene encoding acetylglucosamine deaminase (nagB)

[0025] The plasmid pTSC (Nanjing Agricultural University, gifted by Dr. Yan Xin, NCBI accession no.EU864234) was transformed into recombinant Bacillus subtilis that had knocked out nagP to eliminate bleomycin resistance. Since the gene encoding acetylglucosamine deacetylase (nagA) and the gene encoding acetylglucosamine deaminase (nagB) are in the same operon of the Bacillus subtilis (Bacillus subtilis168) genome, nagA can be knocked out simultaneously by a single homologous recombination and nagB two genes.

[0026] According to the upstream and downstream sequences of the Bacillus subtilis (Bacillus subtilis168) acetylglucosamine deacetylase coding gene (nagA) and acetylglucosamine deaminase coding gene (nagB) published on NCBI, the knockout frame homology arm amplification primers were designed, The left arm upstream and downstream ...

Embodiment 3

[0033]Example 3 Construction of acetylglucosamine-producing recombinant Bacillus subtilis

[0034] According to the glucosamine acetylase encoding gene (GNA1) in Saccharomyces cerevisiae S288C, purchased from the American Type Microorganism Collection, ATCC204508 published on NCBI, the primer GNA1-F was designed:

[0035] 5'-GGGGTACCATTATAGGTAAGAGAGGAATGTACACATGAGCTTACCCGATGGATTTTATA-3', GNA1-R: 5'-CCCAAGCTTCTATTTTCTAATTTGCATTTCCACG-3'. The gene encoding glucosamine acetylase (GNA1) was amplified from the Saccharomyces cerevisiae S288C genome using the above primers. The amplified fragment was digested with KpnI and HindIII and then ligated into the pP43NMK expression vector (kindly provided by Dr. Zhang Xiaozhou, Virginia Tech, USA). Restriction digestion and sequencing confirmed the successful construction of the recombinant plasmid pP43-GNA1.

[0036] The constructed expression vector pP43-GNA1 was transformed into Bacillus subtilis BSGN3. GNA1-F and GNA1-R primers were ...

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Abstract

The invention discloses bacillus subtilis engineering bacteria with high yield of acetylglucosamine and application of the bacillus subtilis engineering bacteria with high yield of the acetylglucosamine. Bacillus subtilisi 168 serves as an original strain. On the basis that homologous recombination knocks out acetylglucosamine glucose transporter protein coding gene (nagP), acetylglucosamine deacetylation enzyme coding gene (nag A) and acetylglucosamine deamination enzyme coding gene (nag B) are further knocked out so as to block up acetylglucosamine degradation pathway in host bacteria. In the host bacteria without the nag A and the nag B, acetylglucosamine acetylase coding gene (GNA1) from Saccharomyces cerevisiae S288C is overly expressed, an acetylglucosamine synthetic route is strengthened to produce recombination bacillus subtilis for producing acetylglucosamine, yield reaches 1.23 grams per liter, and the method is a basis for metabolic engineering to further modify bacillus subtilis to produce glucosamine.

Description

technical field [0001] The invention relates to a high-yielding acetylglucosamine-producing Bacillus subtilis engineering bacterium, in particular to a method for improving the production of recombinant Bacillus subtilis acetylglucosamine by knocking out nagA and nagB through homologous recombination, and belongs to the field of genetic engineering. Background technique [0002] Acetyl glucosamine is a kind of monosaccharide in organisms, which widely exists in bacteria, yeast, mold, plants and animals. In the human body, acetylglucosamine is the synthetic precursor of the disaccharide unit of glycosaminoglycan, which plays an important role in the repair and maintenance of cartilage and joint tissue functions. Therefore, acetyl glucosamine is widely used as a drug and nutritional dietary supplement to treat and repair joint damage. In addition, acetylglucosamine also has many applications in the field of cosmetics and pharmaceuticals. At present, acetyl glucosamine is mai...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12P19/26C12R1/125C12R1/865
Inventor 陈坚堵国成刘龙李江华刘延峰
Owner JIANGNAN UNIV
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